WNT-dependent interaction between inflammatory fibroblasts and FOLR2+ macrophages promotes fibrosis in chronic kidney disease

Chronic kidney disease (CKD) is a public health problem driven by myofibroblast accumulation, leading to interstitial fibrosis. Heterogeneity is a recently recognized characteristic in kidney fibroblasts in CKD, but the role of different populations is still unclear. Here, we characterize a proinflammatory fibroblast population (named CXCL-iFibro), which corresponds to an early state of myofibroblast differentiation in CKD. We demonstrate that CXCL-iFibro co-localize with macrophages in the kidney and participate in their attraction, accumulation, and switch into FOLR2+ macrophages from early CKD stages on. In vitro, macrophages promote the switch of CXCL-iFibro into ECM-secreting myofibroblasts through a WNT/β-catenin-dependent pathway, thereby suggesting a reciprocal crosstalk between these populations of fibroblasts and macrophages. Finally, the detection of CXCL-iFibro at early stages of CKD is predictive of poor patient prognosis, which shows that the CXCL-iFibro population is an early player in CKD progression and demonstrates the clinical relevance of our findings.


April 2023
Randomization For all in vitro and coculture studies, all the fibroblast cell lines available (3) were cocultured in the same time by the same healthy PBMC donors (n=1-2 depending on the day of the experiment).Healthy donors were randomly selected and blind to the investigator.Etablissement Francais du Sang was in charge to collect and deliver blood from healthy donor to Institut Curie Blinding For quantification of immunohistochemistry or immunofluorescence in humans, the investigator was blind from the pathological assessment, and especially the degree of fibrosis For quantification of MFI in vitro, FACS analysis the investigator was blind from treatment administrated to cells.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Study protocol
For the Neptune study, the design can be found on the following reference : Design of the Nephrotic Syndrome Study Network (NEPTUNE) to evaluate primary glomerular nephropathy by a multidisciplinary approach, Gadegbeku et al., DOI 10.1038/ki.2012.428.

Data collection
NEPTUNE is a multicenter observational, prospective cohort study of children and adults with proteinuric glomerular disease, for which comprehensive clinical and molecular phenotyping data was collected at 21 sites at the time of first clinically indicated renal biopsy {Gadegbeku, 2013 #71}.Biospecimens were collected after informed consent and with approval of the local ethics committee {Ju, 2015 #45}.For the patients not in the NEPTUNE cohort (PKD patients, kidney biopsy cohort, spatail transcriptomics): data were pseudoanonymized and data collection was perfromed on a securized excel spreadsheet (password).Data were stored on a securized server at the institut Curie.

Outcomes
Primary oucome was defined as a classical outcome in chronic kidney disease progression studies, such as reach of end-stage renal disease or decrease of eGFR of more than 40%

Novel plant genotypes
Describe the methods by which all novel plant genotypes were produced.This includes those generated by transgenic approaches, gene editing, chemical/radiation-based mutagenesis and hybridization.For transgenic lines, describe the transformation method, the number of independent lines analyzed and the generation upon which experiments were performed.For gene-edited lines, describe the editor used, the endogenous sequence targeted for editing, the targeting guide RNA sequence (if applicable) and how the editor was applied.

Seed stocks
Report on the source of all seed stocks or other plant material used.If applicable, state the seed stock centre and catalogue number.If plant specimens were collected from the field, describe the collection location, date and sampling procedures.

Authentication
Describe any authentication procedures for each seed stock used or novel genotype generated.Describe any experiments used to assess the effect of a mutation and, where applicable, how potential secondary effects (e.g.second site T-DNA insertions, mosiacism, off-target gene editing) were examined.The axis labels state the marker and fluorochrome used (e.g.CD4-FITC).
The axis scales are clearly visible.Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells percentage (with statistics) is provided.

Cell population abundance
The whole populaiton from the cell culture was analyzed

Gating strategy
Gating strategy consisted in selected cells, then singlets using FSC/A-FSC-H, then live cells (live-dead dye).CD14+ CD16+ cells were gated, followed by CD206+ cells.Finally FOLR2+ cells were quantified Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.